Gibberellin 2β-hydroxylase genes of rice and uses thereof

Abstract

Novel GA2β-hydroxylase genes were successfully isolated from rice. In addition, plants whose plant type has been modified compared with their wild type counterparts, were successfully constructed via these genes.

Description

[0001]This application claims the benefit of prior-filed Japanese Patent Application 11 / 365899 (filed Dec. 24, 1999) entitled “Rice Origin Gibberellin 2β-Hydroxylase Genes and Utilization Thereof”. The entire content of the above-referenced application is incorporated herein by reference.TECHNICAL FIELD[0002]The present invention relates to genes of rice involved in gibberellin biosynthesis and uses thereof.BACKGROUND ART[0003]Gibberellins (GAs) form a large family of tetracyclic diterpenoid carboxylic acids that have the basic structure called ent-gibberellane (FIG. 1A). They control multiple processes in the life cycle of higher plants, which are essential for normal plant growth and development (Graebe, J. E. (1987). Ann. Rev. Plant Physiol., 38, 419–465; Hooley, R. (1994). Plant Mol. Biol., 26, 1529–1555). Biologically active GAs, such as GA1, are produced from trans-geranylgeranyl diphosphate mediated by the sequential actions of cyclases in the plastids, membrane-associated mo...

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Benefits of technology

[0039]Furthermore, studies on GA-deficient mutants and actions of exogenous GAs and / or inhibitors applied to plants for GA biosynthesis have revealed that GAs are essential and potent regulators for plant growth. These GAs influence various phenomena in the growth of plants having a relatively high stature, and are also involved in the stimulation of stem elongation. In fact, plants in which the OsGA2ox1 cDNA of the present invention is expressed become severely dwarfed. Therefore, GA 2β-hydroxylase of the present invention and DNA encoding the enzyme may be useful in modifying plant growth, for example, production of a plant whose plant type differs from that of a wild type. Modification of plant type, dwarfing in particular, provides a variety of agronomical advantages such as a high density of planting, efficient photoreception, decrease in wind damage, reduction of farming labor, etc. Dwarfing is thus the most valuable trait for breeding agricultural and horticultural products, including fruit trees.

[0046]As described above, the genes isolated by the present inventors are assumed to be involved in the plant growth through the production of biologically inactive GAs. Therefore, plant growth may be controlled by regulating the expression of these genes. Since these genes in particular are thought to be involved in the internodal growth of plants, this gene may be utilized in the control of plant stature. Control of plant stature provides a variety of industrial advantages. For example, the shortened stature caused by increasing the expression of the gene of this invention in a plant can make the plant resistant to bending thereby increasing the fruit weight. Furthermore, the shortened stature makes the size of the plant per stub more compact so that the number of plants to be planted per unit area can be increased. This dense planting is highly important in the production of agricultural products including rice, wheat, maize, etc., in particular. DNA encoding the GA 2β-hydroxylase of the present invention may be applicable to dwarf flowering plants, dwarf fruit trees, etc.

[0051]An antisense sequence used in the present invention can suppress the target gene expression by any of the above-mentioned mechanisms. If an antisense sequence is designed to be complementary to the untranslated region near the 5′ end of the gene's mRNA; it will effectively inhibit translation of a gene. Additionally, it is also possible to use sequences that are complementary to the coding regions or to the untranslated regions on the 3′ side. Thus, the antisense DNA used in the present invention includes a DNA having antisense sequences against both the untranslated regions and the translated regions of the gene. The antisense DNA to be used is connected downstream from an appropriate promoter, and, preferably, a sequence containing the transcription termination signal is connected on the 3′ side. The DNA thus prepared can be transfected into the desired plant by known methods. The sequence of the antisense DNA is preferably a sequence complementary to the endogenous gene (or the homologue) of the plant to be transformed or a part thereof, but it need not be perfectly complementary so long as it can effectively inhibit the gene expression. The transcribed RNA is preferably not less than 90%, and most preferably not less than 95% complementary to the transcribed products of the target gene. In order to effectively inhibit the expression of the target gene by means of an antisense sequence, the antisense DNA should be at least 15 nucleotides long or more, preferably 100 nucleotides long or more, and most preferably 500 nucleotides long or more. The antisense DNA to be used is generally shorter than 5 kb, and preferably shorter than 2.5 kb.

[0055]The ribozyme designed to cleave the target is fused with a promoter, such as the cauliflower mosaic virus 35S promoter, and with a transcription termination sequence, so that it will be transcribed in plant cells. However, if extra sequences are added to the 5′ end or the 3′ end of the transcribed RNA, the ribozyme activity may be lost. In this case, one can place an additional trimming ribozyme, which functions in the cis position to perform the trimming on the 5′ or the 3′ side of the ribozyme portion, thereby precisely cutting the ribozyme portion from the transcribed RNA containing the ribozyme (Taira, K. et al. (1990). Protein Eng. 3, 733; Dzaianott, A. M. and Bujarski, J. J. (1989). Proc. Natl. Acad. Sci. USA 86, 4823; Grosshands, C. A. and Cech, R. T. (1991). Nucleic Acids Res. 19, 3875; Taira, K. et al. (1991.) Nucleic Acid Res. 19, 5125). Multiple sites within the target gene can be cleaved by arranging these structural units in tandem to achieve greater effects (Yuyama, N. et al., (1992). Biochem. Biophys. Res. Commun. 186, 1271). By using such ribozymes, it is possible to specifically cleave the transcription products of the target gene in the present invention, thereby suppressing the expression of the gene.

Problems solved by technology

However, this method of producing dwarf plants by antisense expression of these active GA-forming enzyme genes has two major defects: 1) it is difficult to predict and regulate an endogenous level of GA, because expression of homologue genes which exist in the same species as the plant, into which the antisense construct will be introduced, may not be suppressed and the half-life of active gibberellins is extended due to the suppression of expression of genes encoding 2β-hydroxydases that produces biologically inactive GAs, and 2) it is necessary to isolate the corresponding cDNA from the same plant species as the plant into which the antisense construct will be introduced.

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